Expression of proteins with dimethylarginines in Escherichia coli for protein–protein interaction studies

Abstract

Protein arginine methylation often modulates protein-protein interactions. To isolate a sufficient quantity of proteins enriched in methyl arginine(s) from natural sources for biochemical studies is laborious and difficult. We describe here an expression system that produces recombinant proteins that are enriched in v-N G ,N G -asymmetry dimethylarginines. A yeast type I arginine methyltransferase gene (HMT1) is put on a plasmid under the control of the Escherichia coli methionine aminopeptidase promoter for constitutive expression. The protein targeted for post-translational modification is put on the same plasmid behind a T7 promoter for inducible expression of His 6 -tagged proteins. Sbp1p and Stm1p were used as model proteins to examine this expression system. The 13 arginines within the arginine-glycine-rich motif of Sbp1p and the RGG sequence near the C terminus of Stm1p were methylated. Unexpectedly, the arginine residue on the thrombin cleavage site (LVPRGS) of the fusion proteins can also be methylated by Hmt1p. Sbp1p and Sbp1p/hmt1 were covalently attached to solid supports for the isolation of interacting proteins. The results indicate that arginine methylation on Sbp1p exerts both positive and negative effects on protein-protein interaction.

Biophysical and biochemical analysis of hnRNP K: arginine methylation, reversible aggregation and combinatorial binding to nucleic acids

“…The recombinant expression of arginine methylated proteins in E. coli has previously been reported for two yeast proteins, Sbp1p and Stm1p, which were co-expressed with yeast type I arginine methyltransferase (Hmt1p) (Hsieh et al, 2007). In our hands, a simple co-expression from a bicistronic plasmid did not result in quantitative hnRNP K methylation.…”

Enhanced Methylarginine Characterization by Post-Translational Modification-Specific Targeted Data Acquisition and Electron-Transfer Dissociation Mass Spectrometry

“…Dense methylation of GAR domains is emerging as a common theme amongst protein arginine methyltransferase (PRMT) substrates [16,48], and the lack of strong enrichment techniques for methylated peptides suggests that recombinant PRMT substrate production is likely to play an important role in the comprehensive localization of these sites [6,49]. As additional PRMT substrates are identified and targeted for methylarginine site localization, the benefits of the abovementioned PTM-specific targeted data acquisition workflow should be particularly marked.…”

Sbp1 modulates the translation of Pab1 mRNA in a poly(A)- and RGG-dependent manner

“…Post-translational modifications of the RGG domain of Sbp1 were known to modulate its molecular interactions in the cell (Hsieh et al 2007). In this study, we demonstrated that arginine methylation in the RGG repeats of Sbp1 compromised the binding of Sbp1 to Pab1.…”

“…S2A; Hsieh et al 2007). We then confirmed methylation of arginines in the RGG domain by mass spectrometry (Supplemental Fig.…”

“…Methylation of the arginine in the RGG domain of Sbp1 was done essentially as previously described (Hsieh et al 2007) with the following modifications. Briefly, plasmids containing Sbp1 and type I arginine methyltransferase Hmt1 were cotransformed and coexpressed in E. coli BL21 (DE3) cells.…”